Cold-adapted Aniline Degrading Bacterium Pseudomonas Migulae AN-1:Aerobic Denitrification And Application For Remediation Of Nitrate Contaminated Groundwater By Bio-circulation Well

With the rapid development of industry and agriculture, discharge of waste water, industrial waste, leaks of municipal landfill leachate and abuse of pesticides and fertilizers leads to heavy groundwater contamination. According to a recent survey of national groundwater from China Geological Survey, the type and quantity of nitrogen compounds in groundwater pollution is rising.Nitrogen compounds, in particular the nitrate pollution, are more realistic to use in-situ remediation. But in most case, PRB is only applicable to shallow groundwater pollution remediation and barrier media is difficult to replace. Groundwater Circulation Wells overcome the defects these problem, and has been successful in terms of volatile contaminants removal. However its effectiveness and applicability in non-volatile contaminants(such as nitrates, heavy metals, etc.) is lack of systematic research.Thinking of the GCW’s efficiently interior space, GCW can combine with some technology on the ground. It’s possible to build a bioreactor by puting the fillers or media, with immobilized denitrify bacteria, into the well, from technology and economic. However the GCW’s circulation is powered by air, the injection of air increase the dissolve oxygen in aquifer that limits the anaerobic denitrification. In addition, in the most case the average temperature of groundwater is about 10℃, it makes the adaptability and denitrification characteristics becoming the key to applicate bio-remdiation of groundwater nitrate pollution. So it’s the primary problem to isolate cold-adapted aerobic denitrifying bacteria.This paper starts at this, first cold-adapted aniline-degrading bacteria is isolated and enriched. Then build GWC bioreactor for remediating nitrate contaminated groundwater, after knowing the mechanism aerobic denitrification in low temperature conditions. It’s aiming at obtaining new strains of denitrifing bacteria, expanding the applicability of GWC and richening nitrate remediation technology and the mechanism of the tech. The operation and research on these economic and efficiency remediation tech will improve the groundwater quality, ensure the citizen’s water safety. It’s important to the social and environment.In the present study, a psychrotrophic bacterium of Pseudomonas migulae AN-1 capable of effectively degrading aniline at low emperatures was screened and characterized. The nitrogen removal and metabolic mechanisms were evaluated at 10 C under aerobic conditions. Response surface methodology(RSM) was applied to optimize the conditions of nitrate removal by AN-1 at low temperature.Genome sequencing and the application of AN-1 for remediation of nitrate contaminated groundwater by Bio-circulation well was also investigated. The main conclusions is following:1) Found a cold-adapted aniline-degrading bacteria AN-1, molecular identification of bacteria is Pseudomonas. It can effectively degrade aniline with concentration between 135~2202 mg/L. Temperature feature shows the optimum growth temperature of AN-1 is 20℃, it can’t grow above 35℃, belongs psychrophilic. AN-1 has a wide range of pH(5~9) adaptability.2) AN-1 has heterotrophic aerobic nitrification and denitrification. At 10 ℃ its removal rates of ammonia, nitrate, nitrite are 1.56, 1.57 and 0.69 mg/(L·h). Nitrate removal at low temperatures than full compliance with substrate inhibition kinetics, the model parameters K = 270.27 mg NO3-Ng/L/h, KS = 43.65 mg/L, and KSI = 185 mg/L. Nitrogen balance analysis shows that AN-1 at low temperature initial ammonia(102 mg/L) of 33.22 percent into nitrogen. Its approach ammonia removal of one is cell assimilation, another is NH4 + â†' NH2 OH â†' NO2- â†' N2 O â†' N2.3) Optimization of response surface method AN-1 degrade nitrate: Nitrate = 65.29 mg/L, initial pH = 6.65, the initial DO = 7.53 mg/L. Model predictive value(53.81%) and actual values(53.75%) are in good fitness, this model has a certain practical significance. The 3D response surface analysis demonstrates that when the pH is close to 7, the concentration of nitrate nitrogen is close to 70mg/L, system can obtain a high removal rate, and when the DO vary between 6-9mg/L, has little effect on the removal rate under aerobic conditions.4)Illumina Miseq sequencing technology was used to determine the genome sequence of Pseudomonas migulae AN-1. Genome size is about 7325406 bp with GC content of 58.92%. The number of cotings is 153. 140 scaffold were produced, of which 19 ORF related denitrification genes were found. Most of the genes are highly homologous. All the four genes of redox enzymes for denitrification similar to those of Pseudomonas fluorescens were identified in Pseudomonas migulae AN-1.5)Bio-GWC working for 26 days, nitrate removal rate has been increased by many S-shaped uniform, sampling points’ highest concentration decreased from 455.32 mg/L to 271.89 mg/L(B8 point). The average concentration in simulated tank decreased from 301.79 mg/L to 110.84 mg/L, removal rate is about 62%. AN-1 on the vinylon fibers, during processing, biomass increased by about 20 times. During the use of bio-GWC, the generation of nitrite is low, so it’s a safe in-situ remediation without secondary pollution.